M/Z targeted attenuation on time of flight instruments

ABSTRACT

A method of mass spectrometry is disclosed comprising separating ions according to one or more physico-chemical properties. Ions which are onwardly transmitted to a Time of Flight mass analyzer are controlled by attenuating ions which would otherwise be transmitted to the Time of Flight mass analyzer and cause saturation of an ion detector and which have been determined or which are predicted to have a relatively high intensity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/004,963, filed 13 Sep. 2013, which is the National Stage ofInternational Application No. PCT/GB2012/050576, filed 15 Mar. 2012,which claims priority from and the benefit of U.S. Provisional PatentApplication Ser. No. 61/452,772 filed on 15 Mar. 2011 and United KingdomPatent Application No. 1104292.6 filed on 15 Mar. 2011. The entirecontents of these applications are incorporated herein by reference.

BACKGROUND TO THE PRESENT INVENTION

This invention relates to apparatus and methods for improving thein-spectrum dynamic range of tandem Time of Flight (“TOF”) massspectrometers. Separation of ions prior to Time of Flight analysis hasmany existing applications.

According to a first example, ions may be separated by gas phasemobility (which in turn depends on shape and charge) allowingelucidation of structural information and/or removal of interference.

According to a second example, ions may be separated by mass to chargeratio (m/z) or mobility prior to fragmentation, reducing interferenceand improving confidence in assignment of fragment ions to precursorions.

According to a third example, as packets of ions of equal energyproduced by a travelling wave device travel into the pusher region of anorthogonal acceleration Time of Flight instrument, the constituent ionsseparate according to their mass to charge ratio. The timing of the Timeof Flight pusher can be adjusted to allow enhancement in duty cycleoptimised at chosen mass to charge ratios.

According to a fourth example, when the packets of ions described in thethird example have been separated by ion mobility, it is possible toadjust the pusher synchronisation independently for each packet. Sincemobility and mass to charge ratio are correlated, this results in anenhancement in duty cycle across the whole mass to charge ratio range.

The fourth example is an example of a High Duty Cycle (or “HDC”) mode ofoperation of an orthogonal acceleration Time of Flight instrument. Forthe purposes of the present application, HDC operation entails at leastone stage of separation and packetisation according to a physicochemicalproperty that is correlated with mass to charge ratio andsynchronisation of the orthogonal acceleration Time of Flight pusher tooptimise transmission of a particular mass to charge ratio value foreach packet.

In many applications, ions are accumulated prior to separation to avoidloss of sensitivity. When the effects of ion accumulation, separationand improved duty cycle are combined for any particular species, themaximum ion current observed at the ion detector can be increasedsubstantially. For low abundance components this results in improvementin the limit of detection, quantification and mass measurement. However,for high abundance species, the resulting ion current can exceed thedynamic range of the ion detector to the detriment of mass measurementand quantification.

Known methods of attenuation of ion signals typically reduce thetransmission of all ions to some extent.

It is desired to provide an improved mass spectrometer and method ofmass spectrometry.

SUMMARY OF THE INVENTION

According to an aspect of the present invention there is provided amethod of mass spectrometry comprising:

separating ions according to one or more physico-chemical properties;

providing a Time of Flight mass analyser; and

controlling ions which are onwardly transmitted to the Time of Flightmass analyser by attenuating first ions having a first physico-chemicalproperty within one or more first ranges which would otherwise betransmitted to the Time of Flight mass analyser and which have beendetermined to have or which are predicted to have a relatively highintensity.

According to the preferred embodiment the first ions are attenuated ifthey are determined or predicted to cause saturation of other adverseaffects to the ion detector.

The step of controlling ions which are onwardly transmitted to the Timeof Flight mass analyser preferably further comprises attenuating firstions having a second physico-chemical property within one or more secondranges.

According to an embodiment a two dimensional or multidimensionalseparation is performed wherein ions are simultaneously separatedaccording to two different physico-chemical properties (e.g. ionmobility and mass to charge ratio) and wherein first ions which areattenuated have both a first physico-chemical property (e.g. ionmobility) within one or more first (e.g. ion mobility) ranges and asecond physico-chemical property (e.g. mass to charge ratio) within oneor more second (e.g. mass to charge ratio) ranges.

According to another embodiment a plurality of one dimensional or singledimensional separations are performed in series or sequentially whereinions are initially separated according to a first physico-chemicalproperty (e.g. ion mobility or mass to charge ratio) and wherein firstions which are attenuated have a first physico-chemical property (e.g.ion mobility or mass to charge ratio) within one or more first (e.g. ionmobility or mass to charge ratio) ranges and wherein the ions are thensubsequently separated according to a second physico-chemical property(e.g. ion mobility or mass to charge ratio) and wherein first ions whichare attenuated have a second physico-chemical property (e.g. ionmobility or mass to charge ratio) within one or more second ranges.

The step of separating ions according to one or more physico-chemicalproperties preferably comprises separating ions according to their ionmobility.

The first ions which are attenuated preferably have ion mobilitieswithin one or more first ion mobility ranges.

The step of separating ions according to one or more physico-chemicalproperties may less preferably comprise separating ions according totheir mass or mass to charge ratio. According to this embodiment, thefirst ions which are attenuated preferably have masses or mass to chargeratios within one or more first mass or mass to charge ratio ranges.

The step of attenuating the first ions preferably comprises onwardlytransmitting 0%, <10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%,70-80%, 80-90% or >90% of first ions having a physico-chemical propertywithin the one or more first ranges.

The step of attenuating the first ions preferably comprises onwardlytransmitting <10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%,70-80%, 80-90% or 90-100% of other ions having a physico-chemicalproperty outside of the one or more first ranges. According to thepreferred embodiment ions having a physico-chemical property outside theone or more first ranges are not substantially attenuated or lesspreferably are attenuated to a lesser degree.

First ions having a physico-chemical property within the one or morefirst ranges are preferably attenuated to a greater relative extent thanother ions having a physico-chemical property outside of the one or morefirst ranges.

The step of controlling ions which are onwardly transmitted to the Timeof Flight mass analyser preferably comprises controlling the timing atwhich an orthogonal acceleration pulse is applied to an orthogonalacceleration electrode into order to orthogonally accelerate ions into atime of flight region of the Time of Flight mass analyser. The timing ofenergising the orthogonal acceleration electrode is preferably arrangedso that the first ions are not orthogonally accelerated and hence arelost to the system.

The step of controlling ions which are onwardly transmitted to the Timeof Flight mass analyser may comprise controlling one or more ion opticallenses arranged upstream of the Time of Flight mass analyser.

The one or more ion optical lenses are preferably arranged and adaptedto control the focusing or defocusing of an ion beam so that in a modeof operation a reduced intensity of ions is onwardly transmitted.

The step of controlling ions which are onwardly transmitted to said Timeof Flight mass analyser may comprise repeatedly switching an ionattenuation device ON and OFF, wherein the duty cycle of the ionattenuation device may be varied in order to control the degree ofattenuation of the ions.

The method may further comprise post-processing mass spectral dataand/or a mass spectrum wherein the intensity of selected mass to chargeratio data and/or one or more mass or mass to charge ratio peaks isincreased to correct for or compensate for the effect of attenuating thefirst ions.

According to another aspect of the present invention there is provided amass spectrometer comprising:

a device arranged and adapted to separate ions according to one or morephysico-chemical properties;

a Time of Flight mass analyser; and

a control system arranged and adapted to control ions which are onwardlytransmitted to the Time of Flight mass analyser by attenuating firstions having a first physico-chemical property within one or more firstranges which would otherwise be transmitted to the Time of Flight massanalyser and which have been determined to have or which are predictedto have a relatively high intensity.

According to the preferred embodiment the first ions are attenuated ifthey are determined or predicted to cause saturation of other adverseaffects to the ion detector.

According to an aspect of the present invention there is provided a massspectrometer comprising:

one or more separation devices capable of separating ions according toone or more of their physicochemical properties;

one or more signal attenuation devices operating on a timescale shorterthan the range of separation times afforded by the one or moreseparation devices;

a Time of Flight mass spectrometer; and

a means of or device for controlling each signal attenuation device sothat one or more selected regions of the available separation space istargeted for attenuation.

According to an aspect of the present invention there is provided amethod of mass spectrometry comprising:

separating ions according to one or more of their physicochemicalproperties;

providing one or more signal attenuation devices operating on atimescale shorter than the range of separation times afforded by the oneor more separation devices;

providing a Time of Flight mass spectrometer; and

controlling each signal attenuation device so that one or more selectedregions of the available separation space is targeted for attenuation.

The present invention addresses the lack of specificity of attenuationof conventional methods.

The preferred embodiment is possible when ions to be injected into aTime of Flight mass analyser are subjected to preliminary separation byany one of (or a combination of) a variety of physical characteristicsC, C′, C″ . . . on a timescale that is longer than that associated withTime of Flight analysis. It is then possible to employ a variety ofsignal attenuation methods operating on a shorter timescale than thefastest separation to selectively suppress signal for analytes with C,C′, C″ . . . near to one or more sets of target values C_(i), C′_(i),C″_(i) . . . without reducing the detected signal for components outsidethe targeted ranges. Alternatively, when a nested multidimensionalseparation is not available, separation and attenuation in eachdimension may be performed sequentially. This may require the use ofmore than one attenuation device. For example, the following steps maybe performed: (i) separation according to some physicochemicalcharacteristic C; (ii) attenuation near one or more target values C_(i);(iii) separation according to some physicochemical characteristic C′;(iv) attenuation near one or more target values C′_(i) and so on. Thiswill result in some attenuation outside the targeted ranges in eachdimension, but regions where the one dimensional ranges overlap will beattenuated most.

The specificity of the attenuation is preferably determined by thequality of the preliminary separations and the speed of the attenuationmechanism. If the attenuation method is quantitative, then data in theaffected range may be rescaled appropriately for display and/or dataanalysis. In a feedback mode of operation, where the composition of theanalyte is changing with time, the range to target may be determinedautomatically using data already collected.

In a preferred embodiment of the present invention, the method comprisesan ion source upstream of a series of accumulation, separation andattenuation devices and a Time of Flight analyser. At least oneseparation device and one attenuation device is required.

A preferred mode of operation is as follows.

Firstly, ions enter from the ion source and pass into an accumulatingdevice.

Secondly, after a period of accumulation, a packet of ions is releasedinto a separation device. Any particular species will emerge from theseparation device according to some probability distribution Pr(T_(SEP)GIVEN C) where T_(SEP) is the time taken to pass through the separationdevice and C is some physicochemical characteristic (or combination ofphysicochemical characteristics) of the ions.

Thirdly, ions then pass through a device that has transmission which canbe controlled on a timescale that is shorter than the range of observedseparation times, allowing transmission of ions to be correlated withtheir separation time. Transmission is reduced at times close toT*_(SEP)(C_(i)) where the C_(i) are characteristic of one or morespecies targeted for attenuation.

Fourthly, in an optional feedback mode, the values C_(i) chosen forattenuation are adjusted with time as the composition of the sampleentering the instrument changes.

Fifthly, finally the transmitted ions pass into the Time of Flightanalyser for mass measurement.

Possible useful physicochemical characteristics C include, but are notlimited to, mass to charge ratio, mass, charge and gas phase ionmobility.

Some examples of separation devices include ion mobility cells, iontraps and scanwave wherein the height of a DC and/or pseudo-potentialbarrier within an ion trap is progressively varied so that ions emergefrom the ion trap in order or reverse order of their mass to chargeratio.

According to the preferred embodiment the attenuation device iscontrolled on a timescale T_(ATT) that is small or short enough topreserve a useful correlation between the physico-chemicalcharacteristic C and the transmission ratio. In one embodiment thedistribution Pr(T_(SEP) GIVEN C) may be peaked near a characteristictime T*_(SEP)(C) with a peak width given by ΔT(C). If T_(ATT)<ΔT(C) thenthe specificity of attenuation will be limited by the width ΔT(C) of theseparation device. In this case, reducing ΔT(C) will result in improvedspecificity of attenuation.

According to an aspect of the present invention there is provided a massspectrometer comprising:

one or more separation devices capable of separating ions according totheir physicochemical properties;

one or more signal attenuation devices operating on a timescale shorterthan the range of separation times afforded by the one or moreseparation devices;

a Time of Flight mass spectrometer; and

a means of or device for controlling each attenuation device so that oneor more selected regions of the available separation space is targetedfor attenuation.

The Time of Flight mass spectrometer preferably comprises an orthogonalacceleration Time of Flight mass spectrometer.

According to an embodiment one or more targeted regions are chosen insuch a way that selected molecular species that have been detectedpreviously are attenuated.

One or more of the separation devices may be preceded by an accumulationdevice.

One or more of the separation devices may separate by mass to chargeratio.

One or more of the separation devices may separate by ion mobility.

One of the separation devices may comprises a travelling wave ionmobility cell wherein one or more transient DC voltages or potentialsare applied to the electrodes of an ion mobility cell in order to causeions to separate according to their ion mobility.

One or more of the separation devices may comprise a step wave devicecomprising an ion guide having two ion paths wherein ions are switchedfrom a first ion path to a second different ion path. The ion guide may,for example, comprise a plurality of electrodes having apertures whereinat least some of the electrodes comprise conjoined electrodes.

One or more of the separation devices preferably comprises an ion trap.

According to an embodiment one of the attenuation devices may comprise aDynamic Range Enhancement (“DRE”) lens.

One of the attenuation devices preferably comprises the ion optics whichare used to transfer ions into the pusher region of an orthogonalacceleration Time of Flight mass spectrometer.

One separation device may comprise the pusher region of an orthogonalacceleration Time of Flight mass spectrometer.

One of the attenuation devices may comprise the pusher region of anorthogonal acceleration Time of Flight mass spectrometer.

The mass spectrometer is preferably operated in a High Duty Cycle(“HDC”) mode of operation.

The High Duty Cycle (“HDC”) calibration is preferably modified toattenuate one or more targeted regions in ion mobility and mass tocharge ratio space.

The High Duty Cycle (“HDC”) calibration is preferably adaptivelymodified to reflect the composition of previously detected speciesentering the mass spectrometer.

The High Duty Cycle (“HDC”) calibration preferably switches among two ormore alternative paths.

The degree of attenuation is preferably recorded in a form that permitsapproximate reconstruction of the signal that would have been observedin the absence of attenuation.

According to an embodiment the mass spectrometer may further comprise:

(a) an ion source selected from the group consisting of: (i) anElectrospray ionisation (“ESI”) ion source; (ii) an Atmospheric PressurePhoto Ionisation (“APPI”) ion source; (iii) an Atmospheric PressureChemical Ionisation (“APCI”) ion source; (iv) a Matrix Assisted LaserDesorption Ionisation (“MALDI”) ion source; (v) a Laser DesorptionIonisation (“LDI”) ion source; (vi) an Atmospheric Pressure Ionisation(“API”) ion source; (vii) a Desorption Ionisation on Silicon (“DIOS”)ion source; (viii) an Electron Impact (“EI”) ion source; (ix) a ChemicalIonisation (“CI”) ion source; (x) a Field Ionisation (“FI”) ion source;(xi) a Field Desorption (“FD”) ion source; (xii) an Inductively CoupledPlasma (“ICP”) ion source; (xiii) a Fast Atom Bombardment (“FAB”) ionsource; (xiv) a Liquid Secondary Ion Mass Spectrometry (“LSIMS”) ionsource; (xv) a Desorption Electrospray Ionisation (“DESI”) ion source;(xvi) a Nickel-63 radioactive ion source; (xvii) an Atmospheric PressureMatrix Assisted Laser Desorption Ionisation ion source; (xviii) aThermospray ion source; (xix) an Atmospheric Sampling Glow DischargeIonisation (“ASGDI”) ion source; and (xx) a Glow Discharge (“GD”) ionsource; and/or

(b) one or more continuous or pulsed ion sources; and/or

(c) one or more ion guides; and/or

(d) one or more ion mobility separation devices and/or one or more FieldAsymmetric Ion Mobility Spectrometer devices; and/or

(e) one or more ion traps or one or more ion trapping regions; and/or

(f) one or more collision, fragmentation or reaction cells selected fromthe group consisting of: (i) a Collisional Induced Dissociation (“CID”)fragmentation device; (ii) a Surface Induced Dissociation (“SID”)fragmentation device; (iii) an Electron Transfer Dissociation (“ETD”)fragmentation device; (iv) an Electron Capture Dissociation (“ECD”)fragmentation device; (v) an Electron Collision or Impact Dissociationfragmentation device; (vi) a Photo Induced Dissociation (“PID”)fragmentation device; (vii) a Laser Induced Dissociation fragmentationdevice; (viii) an infrared radiation induced dissociation device; (ix)an ultraviolet radiation induced dissociation device; (x) anozzle-skimmer interface fragmentation device; (xi) an in-sourcefragmentation device; (xii) an in-source Collision Induced Dissociationfragmentation device; (xiii) a thermal or temperature sourcefragmentation device; (xiv) an electric field induced fragmentationdevice; (xv) a magnetic field induced fragmentation device; (xvi) anenzyme digestion or enzyme degradation fragmentation device; (xvii) anion-ion reaction fragmentation device; (xviii) an ion-molecule reactionfragmentation device; (xix) an ion-atom reaction fragmentation device;(xx) an ion-metastable ion reaction fragmentation device; (xxi) anion-metastable molecule reaction fragmentation device; (xxii) anion-metastable atom reaction fragmentation device; (xxiii) an ion-ionreaction device for reacting ions to form adduct or product ions; (xxiv)an ion-molecule reaction device for reacting ions to form adduct orproduct ions; (xxv) an ion-atom reaction device for reacting ions toform adduct or product ions; (xxvi) an ion-metastable ion reactiondevice for reacting ions to form adduct or product ions; (xxvii) anion-metastable molecule reaction device for reacting ions to form adductor product ions; (xxviii) an ion-metastable atom reaction device forreacting ions to form adduct or product ions; and (xxix) an ElectronIonisation Dissociation (“EID”) fragmentation device; and/or

(g) one or more energy analysers or electrostatic energy analysers;and/or

(h) one or more ion detectors; and/or

(i) one or more mass filters selected from the group consisting of: (i)a quadrupole mass filter; (ii) a 2D or linear quadrupole ion trap; (iii)a Paul or 3D quadrupole ion trap; (iv) a Penning ion trap; (v) an iontrap; (vi) a magnetic sector mass filter; (vii) a Time of Flight massfilter; and (viii) a Wein filter; and/or

(j) a device or ion gate for pulsing ions; and/or

(k) a device for converting a substantially continuous ion beam into apulsed ion beam.

The mass spectrometer may further comprise a stacked ring ion guidecomprising a plurality of electrodes each having an aperture throughwhich ions are transmitted in use and wherein the spacing of theelectrodes increases along the length of the ion path, and wherein theapertures in the electrodes in an upstream section of the ion guide havea first diameter and wherein the apertures in the electrodes in adownstream section of the ion guide have a second diameter which issmaller than the first diameter, and wherein opposite phases of an AC orRF voltage are applied, in use, to successive electrodes.

An ion mobility spectrometer according to the preferred embodiment maycomprise a plurality of electrodes each having an aperture through whichions are transmitted in use. One or more transient DC voltages orpotentials or one or more DC voltage or potential waveforms may beapplied to the electrodes comprising the ion mobility spectrometer inorder to urge ions along the length of the ion mobility spectrometer.

According to the preferred embodiment the one or more transient DCvoltages or potentials or the one or more DC voltage or potentialwaveforms create: (i) a potential hill or barrier; (ii) a potentialwell; (iii) multiple potential hills or barriers; (iv) multiplepotential wells; (v) a combination of a potential hill or barrier and apotential well; or (vi) a combination of multiple potential hills orbarriers and multiple potential wells.

The one or more transient DC voltage or potential waveforms preferablycomprise a repeating waveform or square wave.

An RF voltage is preferably applied to the electrodes of the ionmobility spectrometer and preferably has an amplitude selected from thegroup consisting of: (i) <50 V peak to peak; (ii) 50-100 V peak to peak;(iii) 100-150 V peak to peak; (iv) 150-200 V peak to peak; (v) 200-250 Vpeak to peak; (vi) 250-300 V peak to peak; (vii) 300-350 V peak to peak;(viii) 350-400 V peak to peak; (ix) 400-450 V peak to peak; (x) 450-500V peak to peak; (xi) 500-550 V peak to peak; (xxii) 550-600 V peak topeak; (xxiii) 600-650 V peak to peak; (xxiv) 650-700 V peak to peak;(xxv) 700-750 V peak to peak; (xxvi) 750-800 V peak to peak; (xxvii)800-850 V peak to peak; (xxviii) 850-900 V peak to peak; (xxix) 900-950V peak to peak; (xxx) 950-1000 V peak to peak; and (xxxi) >1000 V peakto peak.

The RF voltage preferably has a frequency selected from the groupconsisting of: (i) <100 kHz; (ii) 100-200 kHz; (iii) 200-300 kHz; (iv)300-400 kHz; (v) 400-500 kHz; (vi) 0.5-1.0 MHz; (vii) 1.0-1.5 MHz;(viii) 1.5-2.0 MHz; (ix) 2.0-2.5 MHz; (x) 2.5-3.0 MHz; (xi) 3.0-3.5 MHz;(xii) 3.5-4.0 MHz; (xiii) 4.0-4.5 MHz; (xiv) 4.5-5.0 MHz; (xv) 5.0-5.5MHz; (xvi) 5.5-6.0 MHz; (xvii) 6.0-6.5 MHz; (xviii) 6.5-7.0 MHz; (xix)7.0-7.5 MHz; (xx) 7.5-8.0 MHz; (xxi) 8.0-8.5 MHz; (xxii) 8.5-9.0 MHz;(xxiii) 9.0-9.5 MHz; (xxiv) 9.5-10.0 MHz; and (xxv) >10.0 MHz.

The ion mobility spectrometer is preferably maintained at a pressureselected from the group comprising: (i) >0.001 mbar; (ii) >0.01 mbar;(iii) >0.1 mbar; (iv) >1 mbar; (v) >10 mbar; (vi) >100 mbar; (vii)0.001-0.01 mbar; (viii) 0.01-0.1 mbar; (ix) 0.1-1 mbar; (x) 1-10 mbar;and (xi) 10-100 mbar.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present invention will now be described, byway of example only, and with reference to the accompanying drawings inwhich:

FIG. 1 shows probability distributions for two different species;

FIG. 2 illustrates an embodiment wherein a nested two dimensionalseparation based on physiochemical properties has been carried out;

FIG. 3 shows targets with attenuation regions;

FIG. 4 illustrates a targeted attenuation mode;

FIG. 5 illustrates an intense ion species which is desired to beattenuated in order to avoid detector saturation in accordance with anembodiment of the present invention;

FIG. 6A shows simulated TDC spectra for two analytes, FIG. 6B showssimulated TDC spectra for the two analytes wherein the signal for bothanalytes has been reduced by a factor of ×10 and FIG. 6C illustrates anembodiment of the present invention wherein one analyte has beenattenuated whereas the other analyte is unattenuated; and

FIG. 7A illustrates attenuation in a High Duty Cycle acquisition mode ofoperation of an IMS-Time of Flight mass spectrometer and shows a mixedpopulation of ions trapped in preparation for ion mobility separation,FIG. 7B shows the ions separated according to their ion mobility, FIG.7C shows the first ion packet having exited the ion mobility device,FIG. 7D shows the second ion packet having been released from the ionmobility device and FIG. 7E shows the third ion packet having beenreleased into the pusher region.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A preferred embodiment of the present invention will now be described.

FIG. 1 shows probability distributions T_(SEP) of two different ionspecies showing different characteristic times and separation widths.Both distributions have been normalised to have unit area. A signalattenuation device may be utilised during the time periodT*_(SEP)(C₁)+/−½ΔT(C₁) with the result that ion species #1 will besuppressed relative to ion species #2. Note that in this case somereduction of the signal for ion species #2 will also be observed due tothe overlap of the two distributions. This effect disappears withimproving separation (i.e. smaller peak widths ΔT).

The separation device may be replaced by a series of separation devicesoperating on ever shorter timescales, resulting in a nestedmultidimensional separation. This results in extra specificity so longas the attenuation device is operated on the timescale of the fastest(and final) separation.

FIG. 2 illustrates an embodiment of the present invention in which anested two dimensional separation based on physiochemicalcharacteristics C and C′ has been carried out. After the second phase ofseparation, ions are in packets that can be labelled by both C and C′and it is possible to target packets with particular values of C and C′for attenuation. This is further illustrated in FIG. 3. FIG. 3 showspoints in black which have been targeted. According to the preferredembodiment attenuation is carried out in the regions defined by thesolid grey areas or ellipses. Species with separation profilesoverlapping the solid grey ellipses such as Species A will be attenuatedto some extent while other species such as Species B will be unaffected.

According to various embodiments different attenuation devices may beused. For example, a Dynamic Range Enhancement (“DRE”) lens may be used.Alternatively, the ion optics used to manipulate ions as they move intoa pusher region of a Time of Flight mass analyser and the pusher regionitself may be used wherein the timing of individual pushes can becontrolled with sufficient accuracy.

Attenuation may be performed between separation devices in which case itis not required that the corresponding separation timescales are nested.

A single physical device may serve more than one of the purposes listedabove. For example, a travelling wave ion mobility separation device maypacketize ions in a form suitable for subsequent separation. Similarly,a Time of Flight pusher can simultaneously act as a mass to charge ratioseparation and attenuation device.

In one mode of operation of the preferred embodiment, a hybrid IonMobility Spectrometry (“IMS”) Time of Flight (“TOF”) instrument may beoperated in a High Duty Cycle (“HDC”) mode. In this mode the timing ofenergising the pusher electrode is adjusted to maximise transmission ata particular mass to charge ratio for packets of a given ion mobility.In normal operation, the mass to charge ratios are chosen to lie along apath in mobility and mass to charge ratio space which allows, forexample, optimisation of transmission for a selected charge state. Sucha path is known as an High Duty Cycle (“HDC”) calibration. Thissituation is illustrated in FIG. 4 in which the mass to charge ratiothat would be chosen for a packet of ions having a given mobility isdefined by the black line. The High Duty Cycle (“HDC”) calibration inthe figure has been selected for optimisation of transmission of singlycharged (1+) species which lie predominantly in the region inside thedashed line.

A targeted attenuation mode is shown in FIG. 5 in which two alternativecalibrations result in attenuation of a singly charged signal in thevicinity of a species with mass and mobility defined by a large blackdot. The calibrations coincide except in the vicinity of the black dotwhere they diverge to pass the species of interest on opposite sides.Many other calibrations are possible, and it is sometimes beneficial toswitch between several different calibrations. Note that factors used todetermine the size of the detour include the quality of the separationand the degree of attenuation required.

In an optional feedback mode of operation, the paths chosen may changewith time to adapt to the composition of the sample currently enteringthe instrument. According to an embodiment calibration paths may detourto avoid several species. Many attenuation devices are at leastpartially quantitative in the sense that the degree of attenuation is atleast approximately known. When such a device is used then it isbeneficial to record the degree of attenuation used so that theunderlying (unattenuated) signal can be at least approximatelyreconstructed.

FIGS. 6A-6C show three simulated TDC spectra for two analytes. The firstanalyte A has a mass to charge ratio of 550 and the second analyte B hasa mass to charge ratio of 748. The two analytes A,B have Electrospray MSresponses which differ by a factor of 10³.

In FIG. 6A no attenuation is used, and the isotope distribution ofanalyte A is severely distorted by detector leadtime.

In FIG. 6B an attenuation device has been employed to reduce the signalfor both analytes A,B by a factor of ×10. This has improved the isotopedistribution for species A, but species B is now so weak that its finalisotope is no longer visible.

FIG. 6C illustrates an embodiment of the present invention whereinspecies A has been targeted for attenuation by a factor of ×10 whilstspecies B is unaffected or unattenuated. This degree of specificity isachievable on current IMS-TOF instruments. The entire isotopedistributions of both species are now recorded faithfully.

FIGS. 7A-E illustrate attenuation according to an embodiment of thepresent invention wherein an IMS-TOF mass spectrometer is operated in aHDC acquisition mode.

FIG. 7A shows a mixed population of ions trapped in preparation for ionmobility separation. Three species are present. The species in black(with intermediate mass to charge ratio and ion mobility) is ofrelatively high abundance and attenuation of this species is desired inorder to prevent saturation of the ion detector.

FIG. 7B shows ions which have been separated into packets according toion mobility. The rightmost packet contains mainly the smallest ionshaving the highest mobility. The central packet contains a mixture ofsmall ions and intermediate mobility ions. The final packet containsintermediate and low mobility ions.

After ions leave the ion mobility device, each packet passes into afield free i.e. a short time of flight region in which the constituentions begin to separate by mass to charge ratio. The timing of a pusherpulse applied to a pusher electrode is preferably adjusted such that,for each packet, ions in a particular mass to charge ratio range arepreferentially pushed into the main time of flight region of the Time ofFlight mass analyser. The variation of pusher timing with mobilityseparation time is referred to as the HDC calibration.

As shown in FIG. 7C, the first ion packet has exited the ion mobilitydevice. The small ions have a lower mass to charge ratio than the ionsof intermediate size and enter the pusher region first. The pusher pulsehas been timed so that the small (and low mass to charge ratio) ions arepushed downwards into the main Time of Flight region, while theintermediate (in size and mobility) ions pass straight through thepusher region and are subsequently discarded.

In FIG. 7D, the second packet has been released from the ion mobilitydevice and the pusher timing has been adjusted such that the small (lowmass to charge ratio) ions and only a small fraction of the intermediateions are pushed into the main Time of Flight region.

In FIG. 7E, the third packet has been released into the pusher region.In this case, the pusher has been timed to transmit the large ions anddiscard the ions of intermediate size and mass to charge ratio.

According to an embodiment the species or regions to be targeted forattenuation may be identified using data already collected in the sameexperiment. For example, during an LC-MS experiment in which more thanone spectrum is acquired during the elution of a chromatographic peak,it is possible to identify (in real time) species with high or risingintensities and to target these for attenuation. Alternatively, data maybe acquired specifically for the purpose of determining attenuationregions. For example, short “pre-scan” acquisitions may be inserted toidentify highly abundant species to target for attenuation. Thispre-scan data may be retained for diagnostic purposes, or simplydiscarded.

Although the present invention has been described with reference to thepreferred embodiments, it will be understood by those skilled in the artthat various changes in form and detail may be made without departingfrom the scope of the invention as set forth in the accompanying claims.

The invention claimed is:
 1. A method of mass spectrometry conductedwith a Time of Flight mass analyser including an ion detector, saidmethod comprising: controlling ions which are transmitted to said Timeof Flight mass analyser by attenuating first ions having a firstphysico-chemical property and a second physico-chemical property withinone or more targeted attenuation regions which would otherwise betransmitted to said Time of Flight mass analyser and which have beendetermined to have or which are predicted to have a relatively highintensity such as to cause saturation of said ion detector.
 2. A methodas claimed in claim 1, wherein a two dimensional or multidimensionalseparation is performed wherein ions are simultaneously separatedaccording to said first and second physico-chemical properties andwherein first ions which are attenuated have both a firstphysico-chemical property within one or more first ranges and a secondphysico-chemical property within one or more second ranges.
 3. A methodas claimed in claim 1, wherein a plurality of one dimensional or singledimensional separations are performed in series or sequentially whereinions are initially separated according to said first physico-chemicalproperty and wherein first ions which are attenuated have a firstphysico-chemical property within one or more first ranges and whereinsaid ions are then subsequently separated according to said secondphysico-chemical property and wherein first ions which are attenuatedhave a second physico-chemical property within one or more secondranges.
 4. A method as claimed in claim 1, wherein said step ofattenuating said first ions comprises onwardly transmitting 0%, <10%,10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or >90%of first ions having a first physico-chemical property within one ormore first ranges.
 5. A method as claimed in claim 1, wherein said stepof attenuating said first ions comprises onwardly transmitting <10%,10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or90-100% of other ions having a first physico-chemical property outsideof one or more first ranges.
 6. A method as claimed in claim 1, whereinsaid first ions having a first physico-chemical property within one ormore first ranges are attenuated to a greater relative extent than otherions having a first physico-chemical property outside of said one ormore first ranges.
 7. A method as claimed in claim 1, wherein said stepof controlling ions which are onwardly transmitted to said Time ofFlight mass analyser comprises controlling the timing at which anorthogonal acceleration pulse is applied to an orthogonal accelerationelectrode in order to orthogonally accelerate ions into a time of flightregion of said Time of Flight mass analyser.
 8. A method as claimed inclaim 1, wherein said step of controlling ions which are onwardlytransmitted to said Time of Flight mass analyser comprises controllingone or more ion optical lenses arranged upstream of said Time of Flightmass analyser.
 9. A method as claimed in claim 8, wherein said one ormore ion optical lenses are arranged and adapted to control the focusingor defocusing of an ion beam so that in a mode of operation a reducedintensity of ions is onwardly transmitted.
 10. A method as claimed inclaim 1, wherein said step of controlling ions which are onwardlytransmitted to said Time of Flight mass analyser comprises repeatedlyswitching an ion attenuation device ON and OFF, wherein the duty cycleof said ion attenuation device may be varied in order to control thedegree of attenuation of said ions.
 11. A method as claimed in claim 1,further comprising post-processing mass spectral data or a mass spectrumwherein the intensity of selected mass or mass to charge ratio data orone or more mass or mass to charge ratio peaks is increased to correctfor or compensate for the effect of attenuating said first ions.
 12. Amethod as claimed in claim 1, wherein said first physico-chemicalproperty or said second physico-chemical property is selected from thegroup consisting of: ion mobility; gas phase ion mobility; charge; massto charge ratio; and mass.
 13. A method as claimed in claim 1, whereinsaid first physico-chemical property is different from said secondphysico-chemical property.
 14. A method as claimed in claim 1, whereinsaid step of attenuating said first ions comprises attenuating saidfirst ions having a first physico-chemical property within one or morefirst ranges and a second physico-chemical property within one or moresecond ranges.
 15. A method as claimed in claim 1, further comprising astep of determining said first ions that have or are predicted to have arelatively high intensity such as to cause saturation of said iondetector.
 16. A method as claimed in claim 1, wherein said one or moretargeted attenuation regions are regions of a separation space.
 17. Amethod as claimed in claim 16, wherein said separation space is twodimensional or multidimensional.
 18. A method as claimed in claim 1,wherein said one or more targeted attenuation regions are substantiallyelliptical.
 19. A mass spectrometer comprising: a Time of Flight massanalyser including an ion detector; and a control system arranged andadapted to control ions which are transmitted to said Time of Flightmass analyser by attenuating first ions having a first physico-chemicalproperty and a second physico-chemical property within one or moretargeted attenuation regions which would otherwise be transmitted tosaid Time of Flight mass analyser and which have been determined to haveor which are predicted to have a relatively high intensity such as tocause saturation of said ion detector.